Vcam 1

Anti-mouse and anti-rabbit IgG-conjugated horseradish peroxidase and rabbit polyclonal antibodies specific for fractalkine/CX3CL1, CX3CR1, ICAM-1, VCAM-1, p-PI3K p85α, PI3K p85α, p-Akt, Akt, p-IKKα/β, IKKα/β, p-IκBα, IκBα, p-p65, p65, and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Recombinant human fractalkine/CX3CL1 was purchased from PeproTech (Rocky Hill, NJ, USA). The short hairpin RNA (shRNA) plasmid used for gene knockdown was purchased from the National RNAi Core Facility Platform (Taipei, Taiwan). All siRNAs used were ON-TARGETplus siRNAs and purchased from Dharmacon Research (Lafayette, CO, USA). All other chemicals were obtained from Sigma–Aldrich (St. Louis, MO, USA).

Serial aortic root cryosections were stained with c-kit (CST), sca-1 (Abcam), ICAM-1 (Santa Cruz), and VCAM-1 (Santa Cruz). Images were captured using a fluorescent microscope (× 40).

Western blot was performed using testicular tissue as described in our previous study [33 (link)]. The primary antibodies included anti-activating transcription factor 4 (ATF4, Cell Signaling Technology, Danvers, MA, USA, 1 : 1000), anti-Bcl-2-associated X protein (Bax, Cell Signaling Technology, 1 : 1000), anti-B-cell lymphoma 2 (Bcl-2, Santa Cruz Biotechnology, 1 : 2000), anti-binding immunoglobulin protein (BIP, Cell Signaling Technology, 1 : 1000), anti-caspase12 (Cell Signaling Technology, 1 : 1000), anti-cleaved caspase3 (c-caspase3, Cell Signaling Technology, 1 : 1000), anti-C/EBP homologous protein (CHOP, Cell Signaling Technology, 1 : 1000), anti-GAPDH (Santa Cruz Biotechnology, 1 : 2000), anti-histone H3 (Santa Cruz Biotechnology; 1 : 1000), anti-intercellular adhesion molecule 1 (ICAM-1, Santa Cruz Biotechnology, 1 : 500), anti-inducible nitric oxide synthase (iNOS, Cell signaling Technology, 1 : 1000), anti-NRF2 (Santa Cruz Biotechnology, 1 : 1000), anti-pro-caspase3 (Cell Signaling Technology, 1 : 1000), and anti-vascular cell adhesion molecule 1 (VCAM-1, Santa Cruz Biotechnology, 1 : 500).

Human brain metastasis and glioblastoma biopsies were processed into formalin-fixed and paraffin embedded specimens and sectioned at 6 μm. Tissue sections were deparaffinized and rehydrated, then immunohistochemically stained for VCAM-1 (20 μg/100 uL catalog no. sc8304; Santa Cruz Biotechnology). Adjacent sections were stained for tumor-specific markers: anti-cytokeratin purified clone CAM5.2 (12.5 μg/mL catalog no. 345779; BD Biosciences) for breast cancer and lung adenocarcinoma, and anti-melan A (1:100 catalog no. ab5106; Abcam) for melanoma. Additional sections were stained with anti-CD34 antibody (1:50 catalog no. M7165; Dako) for vascular characterization. In the case of the glioblastoma specimens, insufficient tissue was available for tumor-specific or blood vessel staining. For detailed information on histologic analysis, see Supplementary Materials and Methods.

Brachiocephalic arteries were post-fixed in 10% formalin, embedded in paraffin and sectioned at 5μm intervals. Sections adjacent to the point of maximum vessel occlusion were used for the comparison of plaque composition. Immunohistochemistry was performed using the Vectastain ABC kit (Vector Laboratories, Burlingame, CA, USA). The tissue was first incubated with primary antibodies to IL-17, Foxp3 and VCAM-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C. The sections were counterstained with hematoxylin. Samples were photographed with an Olympus photomicroscope (Tokyo, Japan).